Number of the records: 1

Pollen and Pollen Tube Biology

SYSNO ASEP	0541115
Document Type	M - Monograph Chapter
R&D Document Type	Monograph Chapter
Title	Isolation of the pistil-stimulated pollen tube secretome
Author(s)	Hafidh, Said (UEB-Q)_{RID, ORCID} Honys, David (UEB-Q)_{RID, ORCID}
Number of authors	2
Source Title	Pollen and Pollen Tube Biology, Methods in Molecular Biology. - New York : Humana Press, 2020 / Geitmann A. - ISSN 1064-3745 - ISBN 978-1-0716-0671-1
Pages	s. 41-72
Number of pages	32 s.
Number of copy	2400
Number of pages	332
Publication form	Print - P
Language	eng - English
Country	US - United States
Keywords	Cell–cell communication ; Glycosylation ; lc-ms/ms ; Pollen tube guidance ; Protein secretion ; Sexual plant reproduction
Subject RIV	EB - Genetics ; Molecular Biology
OECD category	Biochemical research methods
R&D Projects	LM2018129 GA MŠMT - Ministry of Education, Youth and Sports (MEYS)
	GA17-23203S GA ČR - Czech Science Foundation (CSF)
	GA18-02448S GA ČR - Czech Science Foundation (CSF)
	GA18-07027S GA ČR - Czech Science Foundation (CSF)
	EF16_019/0000738 GA MŠMT - Ministry of Education, Youth and Sports (MEYS)
Institutional support	UEB-Q - RVO:61389030
UT WOS	000680070700005
EID SCOPUS	85086424545
DOI	10.1007/978-1-0716-0672-8_4
Annotation	Detection of secreted proteins and peptides during pollen tube guidance has been impeded due to lack of techniques to capture the pollen tube secretome without contamination from the female secreted proteins. Here we present a protocol to detect tobacco pollen tube secreted proteins, semi-in vivo pollen tube secretome assay (SIV-PS), following pollen tube crosstalk with the female reproductive tissues. This method combines the advantages of in vivo pollen tube–pistil interaction and filter-aided sample preparation (FASP) techniques to obtain an in-depth proteome coverage. The SIV-PS method is rapid, efficient, inexpensive, does not require specialized equipment or expertise, and provides a snapshot of the ongoing molecular interplay. We show that the secretome obtained is of greater purity (<,1.4% ADH activities) and that pollen tubes are physiologically and cytologically unaffected. A compendium of quality controls is described and a rough guide on downstream bioinformatics analysis is outlined. The SIV-PS method is applicable to all studies of protein secretion using pollen tube as a model and can be easily adapted to other flowering species with modification. The overall duration for this protocol is approximately 8 hours spanning 4 days (an average of 2 h/day per two workers) excluding microscopy and LC-MS/MS analysis.
Workplace	Institute of Experimental Botany
Contact	David Klier, knihovna@ueb.cas.cz, Tel.: 220 390 469
Year of Publishing	2021
Electronic address	http://doi.org/10.1007/978-1-0716-0672-8_4

Number of the records: 1